In recent years the diagnostic arts have come increasingly to employ and rely upon the detection of minute quantities of physiologically important substances innately or adventitiously present in the body. Many such assays involve competition for a limited number of binding sites on specific binding proteins such as antibodies. These techniques have proven both more sensitive and less cumbersome than earlier resorts such as colorimetry and fluorometry. For example, in the last decade or so radioimmunoassay has proven a tool of exquisite sensitivity and specificity in assays for hormones and other substances, permitting their detection in levels ranging down to fractions of a nanogram per milliliter. Commonly, radioimmunoassay involves competition of radioactively labeled antigen and native antigen for antibody, whereafter bound or unbound label is counted and native antigen substractively quantitated. In like manner, other specific binding proteins such as thyroid binding globulin and estradiol receptor protein have been employed in radiometric determinations of their substrates. Despite the capabilities which commend them, radiommunoassay and related radiometric determinations of physiologically important substances leave much to be desired on several fronts. For example, counting equipment is expensive, isotopes may be hazardous to health and are in any event plagued by undesirably short half-lives, free and bound substrate must be separated before counting, the radioactively labeled antigen in some cases differs in specificity from the untagged target of the assay, and the technique is not as amenable to full automation as might be desired. In some quarters, recognition of these disabilities has focused attention on the so-called "chemically modified bacteriophage assay" first reported by Makela, Immunology, 10, 81 (1966) and independently by Haimovich and Sela, J. Immunology, 97, 338 (1966).
A bacteriophage is a bacteriolytic nucleoprotein which, like any virus, injects its genetic content into its host (in the case of bacteriophages, a bacterial cell), and, arrogating the replicative mechanism of the cell to its own purpose, multiplies within the cell, ultimately causing the cell to burst or undergo "lysis." Upon lysis, myriad new bacteriophages are released to infect other bacteria, continuing the process of proliferation. The extent of cell lysis can be visually estimated by counting plaques which are seen in a culture of host bacteria to result from localized proliferation of the bacteriophage. The number of plaques can be related to the concentration of viable phage initially present in the culture medium. At the outset, this led workers to employ plaque counting to quantitate antibody to virous bacteriophages, since the level of phage antibody present could be inversely related to the number of plaques formed by phage which survived inactivation by the antibody. The contribution of Haimovich and Sela, and of Makela, was to recognize that bacteriophage assay could be generalized by attaching immunospecific moieties to the phage, which upon immunological recognition by antibodies specific to those moieties result in phage inactivation. Accordingly, antigen under assay could be made to compete with antigen conjugated to phage for limited binding sites on antibody, and the assay target quantitated by the plaque counting method. In practice, the "chemically modified bacteriophage assay" has proven to be of general application. Thus, bacteriophage assay with a plaque counting endpoint has been employed to assay hormones such as angiotensin and insulin, proteins such as IgG, nucleic acids such as transfer RMA and DMA, enzymes such as ribonuclease and lysozyme, as well as a diverse family of haptens, such as acetyl salicylyl, penicilloyl, prostaglandins, and various nucleosides and nucleotides. In the main, such work has focused on immunological binding, but it is apparent that the bacteriophage assay may employ specific binding proteins other than antibodies, viz., circulating binding proteins and tissue macromolecules which act as receptor proteins. The bacteriophage assay described thus far provides significant advantages not common to the radioimmunoassay technique now in widespread use. The former assay does not require sophisticated counting equipment, no health hazard arises from the use of radioactive materials, unbound substrates need not be separated from bound substrates prior to quantitation, and the participants in the binding reactions enjoy substantially greater shelf lives than do the isotopes employed in radioimmunoassay. Despite the advantage conferred by bacteriophage assay in these regards, and in spite of the fact that bacteriophage assay is generally as sensitive as radioimmunoassay, the latter technique has remained the procedure of choice, it is believed, because of the strictures imposed by the plaque counting method heretofore universally employed with bacteriophage assay. Thus, while bacteriophage assay enjoys the same sensitivity and specificity which has elevated radioimmunoassay above other less cumbersome methods, that endpoint has proven a barrier to its widespread use in the diagnostic arts. This invention addresses that difficulty and, it is believed, solves it.
According to the practice of this invention, the bacteriophage assay is practiced as before through cell lysis. At that point, the assay of the invention diverges from past practice insofar as its endpoint is concerned. Thus, rather than count photolucencies in cell cultures, by my invention the assay target is quantitated by determined intracellular constituents, e.g., enzymes, emitted upon phage induced lysis. The value so determined is compared to one or more like values similarly obtained upon bacteriophage assay of standard containing known quantities of the substance under assay. The determination of one or another intracellular constituent admist of analytic techniques at least as sensitive as plaque count. But the manifold advantages of this invention derive from the greater ease and speed with which those techniques can be implemented. These and other advantages of the invention and the manner in which they are obtained will be clear from the detailed description which follows.